The following includes information that may be useful in understanding the present inventions. It is not an admission that any of the information provided herein is prior art, or material, to the presently described or claimed inventions, or that any publication or document that is specifically or implicitly referenced is prior art.
The present invention relates to, but is not limited to, the fields of microbiology and microbial genetics. The invention relates, for example, to novel bacterial strains, novel nucleotide sequences, novel amino acid sequences, and processes for employing these bacterial strains, novel nucleotide sequences, and/or novel amino acid sequences for fermentative production of amino acids including, but not limited to, L-threonine, L-methionine, L-lysine, L-homoserine, and L-isoleucine. Preferably L-threonine is produced. The invention also relates to the production of animal feed additives. The invention also relates to fermentation and synthesis of fine chemicals including but not limited to those listed above.
In Escherichia coli, the amino acids L-threonine, L-isoleucine, L-lysine and L-methionine derive all or part of their carbon atoms from aspartate (aspartic acid) via the following common biosynthetic pathway (G. N. Cohen, “The Common Pathway to Lysine, Methionine and Threonine,” pp. 147-171 in Amino Acids: Biosynthesis and Genetic Regulation, K. M. Herrmann and R. L. Somerville, eds., Addison-Wesley Publishing Co., Inc., Reading, Mass. (1983)):

The first reaction of this common pathway is catalyzed by one of three distinct aspartate kinases (AK I, II, or III), each of which is encoded by a separate gene and differs from the others in the way its activity and synthesis are regulated.
Aspartate kinase I, for example, is encoded by thrA, its activity is inhibited by threonine, and its synthesis is repressed by threonine and isoleucine in combination. AK II, however, is encoded by metL and its synthesis repressed by methionine (although its activity is not inhibited by methionine or by paired combinations of methionine, lysine, threonine and isoleucine (F. Falcoz-Kelly et al., Eur. J. Biochem. 8:146-152 (1969); J. C. Patte et al., Biochim. Biophys. Acta 136:245-257 (1967)). AK III is encoded by lysC, and its activity and synthesis are inhibited and repressed, respectively, by lysine.
Aspartate semialdehyde dehydrogenase is encoded by the asd gene, and its activity is regulated through multivalent repression by lysine, threonine, and methionine (K. Haziza et al., EMBO J. 1(3):379-384 (1982)). Its synthesis is also reported to be regulated through repression by glucose-6-phosphate (G6P), and through metabolic regulation by guanosine 5′-diphosphate,3′-diphosphate. Aspartate semialdehyde dehydrogenase catalyzes conversion of L-aspartyl-4-P to L-aspartate-semialdehyde (Chassagnole et al., Biochem. J. 356:415-423 (2001)).
Map positions and nucleotide numbers for various genes in the E. coli strain K-12, as reported in Keseler, I. M., et al., Ecocyc: A Comprehensive Database Resource for E. coli, Nucleic Acids Res. 33:D334-7 (2005) are shown below. Of course, this information is provided as illustrative only, and should not be read to limit the claims unless explicitly stated therein.
GeneStart NucleotideEnd Nucleotideasd3,572,9013,571,798thrA3372,799thrB28013733thrC37345020metL41278584130290lysC42312564229907
Two of the AKs, I and II, are not reported to be distinct proteins, but rather a domain of a complex enzyme that includes homoserine dehydrogenase I or II, respectively, each of which catalyzes the reduction of aspartate semialdehyde to homoserine (P. Truffa-Bachi et al., Eur. J. Biochem. 5:73-80 (1968)). Homoserine dehydrogenase I (HD I) is also encoded by thrA; its synthesis is repressed by threonine plus isoleucine, and its activity is inhibited by threonine. Homoserine dehydrogenase II (HD II) is similarly encoded by metL, and its synthesis is repressed by methionine.
Threonine biosynthesis includes the following additional reactions:Homoserine→Homoserine Phosphate→Threonine
The phosphorylation of homoserine is catalyzed by homoserine kinase, a protein which is composed of two identical 29 kDa subunits encoded for by thrB and whose activity is inhibited by threonine (B. Burr et al., J. Biochem. 62:519-526 (1976)). The final step, the complex conversion of homoserine phosphate to L-threonine is catalyzed by threonine synthase, a 47 kDa protein encoded for by thrC (C. Parsot et al., Nucleic Acids Res. 11:7331-7345 (1983)).
Isoleucine can be produced in E. coli using threonine as a precursor (see Hashiguchi et al., Biosci. Biotechnol. Biochem. 63:672-679 (1999). More specifically, isoleucine is produced via the following reactions:Threonine→α-Ketobutyrate→α-Aceto-α-Hydroxybutyrate→α,β-Dihydroxy-β-Methylvalerate→α-Keto-β-Methylvalerate→Isoleucine.
These reactions are said to be catalyzed in E. coli, respectively, by the following enzymes: threonine deaminase (ilvA); aceto-hydroxyacid synthetase I, II, or III (ilvBN, ilvGM, and ilvIH, respectively); dihydroxyacid reductoisomerase (ilvC); dihydroxyacid dehydratase (ilvD); and transaminase-B (ilvE).
The thrA, thrB and thrC genes all belong to the thr operon, a single operon located at 0 minutes on the genetic map of E. coli (J. Thze and I. Saint-Girons, J. Bacteriol. 118:990-998 (1974); J. Theze et al., J. Bacteriol. 117:133-143 (1974)). These genes encode, respectively, for aspartate kinase I, homoserine dehydrogenase I, homoserine kinase and threonine synthase. Biosynthesis of these enzymes is subject to multivalent repression by threonine and isoleucine (M. Freundlich, Biochem. Biophys. Res. Commun. 10:277-282 (1963)).
A regulatory region is found upstream of the first structural gene in the thr operon and its sequence has been reported (J. F. Gardner, Proc. Natl. Acad. Sci. USA 76:1706-1710 (1979)). A thr attenuator, downstream of the transcription initiation site, contains a sequence encoding a leader peptide; this sequence includes eight threonine codons and four isoleucine codons. The thr attenuator also contains the classical mutually exclusive secondary structures that permit or prevent RNA polymerase transcription of the structural genes in the thr operon, depending on the levels of the charged threonyl- and isoleucyl-tRNAs.
Because of the problems associated with obtaining high levels of amino acid production via natural biosynthesis (e.g., repression of the thr operon by the desired product), bacterial strains have been produced having plasmids containing a thr operon with a thrA gene that encodes a feedback-resistant enzyme. With such plasmids, L-threonine has been produced on an industrial scale by fermentation processes employing a wide variety of microorganisms, such as Brevibacterium flavum, Serratia marcescens, and E. coli. 
For example, the E. coli strain BKIIM B-3996 (Debabov et al., U.S. Pat. No. 5,175,107), which contains the plasmid pVIC40, purportedly makes about 85 g/L in 36 hr. The host is a threonine-requiring strain because of a defective threonine synthase. In BKIIEM B-3996, it is the recombinant plasmid, pVIC40, that provides the crucial enzymatic activities, i.e., a feedback-resistant AK I-HD I, homoserine kinase and threonine synthase, needed for threonine biosynthesis. This plasmid also complements the host's threonine auxotrophy.
E. coli strain 29-4 (E. Shimizu et al., Biosci. Biotech. Biochem. 59:1095-1098 (1995)) is another reported example of a recombinant E. coli threonine producer. Strain 29-4 was constructed by cloning the thr operon of a threonine-over-producing mutant strain, E. coli K-12 (PIM-4) (derived from E. coli strain ATCC Deposit No. 21277), into plasmid pBR322, which was then introduced into the parent strain (K. Wiwa, et al., Agric. Biol. Chem. 47:2329-2334 (1983)). Strain 29-4 purportedly produces about 65 g/L of L-threonine in 72 hr.
Similarly constructed recombinant strains have been reported using other organisms. For example, the Serratia marcescens strain T2000 contains a plasmid having a thr operon that reportedly encodes a feedback-resistant thrA gene product and produces about 100 g/L of threonine in 96 hrs (M. Masuda et al., Applied Biochem. Biotechn. 37:255-262 (1992)). All of these strains are said to contain plasmids having multiple copies of the genes encoding the threonine biosynthetic enzymes, which allows over-expression of these enzymes. This over-expression of the plasmid-borne genes encoding threonine biosynthetic enzymes, particularly a thrA gene encoding a feedback-resistant AK I-HD I, reportedly enables these strains to produce large amounts of threonine. Other examples of plasmid-containing microorganisms are reported, for example, in U.S. Pat. Nos. 4,321,325; 4,347,318; 4,371,615; 4,601,983; 4,757,009; 4,945,058; 4,946,781; 4,980,285; 5,153,123; and 5,236,831.
Non-plasmid containing microorganisms have also been reported as threonine producers. Strains of E. coli such as H-8460, which is obtained by a series of conventional mutagenesis and selection for resistance to several metabolic analogs makes about 75 g/L of L-threonine in 70 hours (Kino, et al., U.S. Pat. No. 5,474,918). Strain H-8460 does not carry a recombinant plasmid and has one copy of the threonine biosynthetic genes on the chromosome. The lower productivity of this strain compared to the plasmid-bearing strains, such as BKIIM B-3996, is believed to be due to lower enzymatic activities (particularly those encoded by the thr operon) as these non-plasmid containing strains carry only a single copy of threonine biosynthetic genes.
An L-threonine producing strain of E. coli, KY10935, produced by multiple rounds of mutation, is reported in K. Okamoto, et al., Biosci. Biotechnol. Biochem. 61:1877-1882 (1997). When cultured under optimal conditions with DL-methionine, strain KY10935 is reported to produce as much as 100 g/liter L-threonine after 77 hours of cultivation. The level of L-threonine produced is believed to result from the inability of this strain to take up L-threonine that accumulates extracellularly, resulting in a decrease in the steady-state level of intracellular L-threonine and the release of the remaining regulatory steps in the L-threonine production pathway from feedback inhibition.
Other examples of non-plasmid containing microorganisms are reported, for example, in U.S. Pat. Nos. 5,939,307; 5,474,918; 5,264,353; 5,164,307; 5,098,835; 5,087,566; 5,077,207; 5,017,483; 4,463,094; 3,580,810; and 3,375,173.
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Although the applicants do not wish to be bound by any particular theory, it is believed that in both the non-plasmid and plasmid containing strains of E. coli, the thr operon is controlled by the particular strain's respective native threonine promoter. As described above, the expression of the native promoter is regulated by a mechanism controlled by a region of DNA that encodes a leader peptide and contains a number of threonine and isoleucine codons. This region is translated by a ribosome which senses the levels of threoninyl-tRNA and isoleucinyl-tRNA. When these levels are sufficient for the leader peptide to be translated, transcription is prematurely terminated, but when the levels are insufficient for the leader peptide to be translated, transcription is not terminated and the entire operon is transcribed, which, following translation, results in increased production of the threonine biosynthetic enzymes. Thus, when threonyl-tRNA and/or isoleucinyl-tRNA levels are low, the thr operon is maximally transcribed and the threonine biosynthetic enzymes are maximally made.
In the E. coli threonine-producing strain BKIIM B-3996, the threonine operon in the plasmid is controlled by its native promoter. As a result, the thr operon is only maximally expressed when the strain is starved for threonine and/or isoleucine. Since starvation for threonine is not possible in a threonine-producing strain, these strains have been rendered auxotrophic for isoleucine in order to obtain a higher level of enzymatic activity.
Another way of overcoming attenuation control is to lower the level(s) of threonyl-tRNA and/or isoleucinyl-tRNA in the cell. A thrS mutant, for example, having a threonyl-tRNA synthase which exhibits a 200-fold decreased apparent affinity for threonine, results in over-expression of the thr operon, presumably due to the low level of threonyl-tRNA (E. J. Johnson, et al., J. Bacteriol., 129:66-70 (1977)).
In fermentation processes using these strains, however, the cells must be supplemented with isoleucine in the growth stage because of their deficient isoleucine biosynthesis. Subsequently, in the production stage, the cells are deprived of isoleucine to induce expression of the threonine biosynthetic enzymes. A major drawback, therefore, of using native threonine promoters to control expression of the threonine biosynthetic enzymes is that the cells must be supplemented with isoleucine.
E. coli strains have recently been reported that contain chromosomally integrated thr operons under the regulatory control of a non-native promoter (Wang, et al., U.S. Pat. No. 5,939,307, the entire disclosure of which is incorporated herein by reference). One of these strains, ADM Kat 13, was reported to produce as much as 102 g/L of L-threonine after 48 hours in culture. E. coli strains have also been reported that are produced by inserting in the chromosome of an E. coli cell at least one threonine operon operably linked to a non-native promoter to produce a parent strain, followed by performing at least one cycle of mutagenesis on the parent strain, followed by screening the mutagenized cells to identify E. Coli that produce specified amounts of L-threonine (United States Published Application No. US2002/0106800 A1, to Liaw et al.).
There remains a need in the art for microorganism strains that are culturable and produce amounts of amino acids such as threonine and isoleucine.